過往文獻中學者們使用不同的方式如將化合物與金屬結合，形成環化錯合物，增強結構的剛性；直接在化合物上接上剛性結構或是降低測量時的溫度等方式來提高綠色螢光蛋白的螢光量子產率。而過去本實驗室的黃世昱學長在研究中意外發現銅離子與o-ABDI在常溫下進行雜環合成反應，提供另一種增加螢光強度的方法。
在本文中接續學長的實驗發展此一鍋化合環反應，藉由改變imidazole上的官能基與銅離子試劑進行反應，驗證其普遍性，之後利用自由基捕捉劑TEMPO探討合環反應中產生的額外產物，然而卻在實驗中發現另一種合環反應，之後我們也對此反應進行推測。
此外，我們將得到的兩種環化產物與環化前產物進行光譜分析，發現環化產物皆螢光量子產率皆有提高。而在imidazole的苯環上做推拉電子基的取代，能觀察到其影響到共軛體系中其他原子上的電子分佈，但對增強螢光效率的影響不大。

Previously, researchers have explored various methods to enhance the fluorescence quantum yield of green fluorescent protein (GFP) analogues, including complexing the compounds with metals to form rigid cyclized complexes, directly attaching rigid structures to the compounds, or lowering the measurement temperature. In a serendipitous discovery, Shih-Yu Huang, a former member of our laboratory, found that copper ions reacted with o-ABDI at room temperature to undergo a heterocyclic cyclization reaction, offering a novel method to increase fluorescence intensity.
Building upon Shih-Yu Huang's earlier work, this study further develops this one-pot cyclization reaction. We investigated the universality of this reaction by modifying the functional groups on the imidazole ring and reacting them with copper ion reagents. Subsequently, we utilized the radical scavenger TEMPO to investigate additional byproducts generated during the cyclization. During this experimental process, we unexpectedly discovered an alternative cyclization reaction, which we then proceeded to hypothesize about.
Furthermore, we conducted spectroscopic analyses on the two resulting cyclized products and their pre-cyclization precursors. Our findings indicate that both cyclized products exhibited enhanced fluorescence quantum yields. While the substitution of electron-donating or electron-withdrawing groups on the phenyl ring of the imidazole was observed to influence the electron distribution on other atoms within the conjugated system, this had only a modest impact on enhancing fluorescence efficiency.

[1] Prasher, D. C., et al., Primary structure of the Aequorea victoria green-fluorescent protein. Gene, 1992, 111, 229 –233 .
[2] Shimomura, O., Structure of the chromophore of Aequorea green fluorescent protein. FEBS Lett. 1979, 104, 220-222.
[3] Chris W. C., et al., Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein. Biochemistry, 1993, 32, 1212-1218
[4] Timothy D. C., Green fluorescent protein: structure, folding and chromophore maturation. Chem. Soc. Rev. 2009, 38, 2865–2875.
[5] Stafforst, T., Diederichsen, U., Synthesis of Alaninyl and N-(2-Aminoethyl)glycinyl Amino Acid Derivatives Containing the Green Fluorescent Protein Chromophore in Their Side Chains for Incorporation into Peptides and Peptide Nucleic Acids. Org. Chem. 2007, 899-911
[6] Lee, C.Y., et al., Facile synthesis of 4-arylidene-5-imidazolinones as synthetic analogs of fluorescent protein chromophore. Tetrahedron. 2012, 68, 5898-5907
[7] Muselli, M., et al., Mild, Efficient, One-Pot Synthesis of Imidazolones Promoted by N,O-Bistrimethylsilylacetamide (BSA). Synlett 2016, 20, 2819-2825
[8] Tsien, R. Y., The green fluorescent protein. Annu. Rev. Biochem. 1998, 67, 509–544
[9] Wu, L. X., Burgess, K., Syntheses of Highly Fluorescent GFP-Chromophore Analogues. J. Am. Chem. Soc. 2008, 130, 12, 4089–4096
[10] Hsu, Y. H., et al., Locked ortho- and para-Core Chromophores of Green Fluorescent Protein; Dramatic Emission Enhancement via Structural Constraint. J. Am. Chem. Soc. 2014, 136, 33, 11805–11812
[11] Chatterjee, S., Ahire, K., Karuso, P., Room-Temperature Dual Fluorescence of a Locked Green Fluorescent Protein Chromophore Analogue. J. Am. Chem. Soc. 2020, 142, 2, 738–749
[12] 陳怡慧, 綠色螢光蛋白類似物的合成、光物理及光異構化反應之探討. 2012, 碩士論文, 國立成功大學化學系.
[13] 黃世昱. 具有鄰位氨基丙烷-2,3-二醇側鏈的綠色螢光蛋白發光團: 合成、性質及應用. 2021, 碩士論文, 國立成功大學化學系.
[14] Zhu, X. Y., et al., Copper-Catalyzed Oxidative Cyclization of Alkynes with Sulfonylhydrazides Leading to 2-Sulfonated 9H-pyrrolo[1,2-a]indol-9-ones. J. Org. Chem. 2017, 82, 16, 8761–8768
[15] Evans, D. A., et al., Chiral Bis(oxazoline)copper(II) Complexes as Lewis Acid Catalystsfor the Enantioselective Diels-Alder Reaction. J. Am. Chem. Soc. 1999, 121, 7559-757
[16] Bailey, W. F., Bobbitt, J. M., Wiberg, K. B., Mechanism of the Oxidation of Alcohols by Oxoammonium Cations. J. Org. Chem. 2007, 72, 12, 4504–4509
[17] 簡翊婷. 銅離子引發綠色螢光蛋白發光團類似物環化反應:副反應探討. 2024, 碩士論文, 國立成功大學化學系.
[18] Kochi, J. K., Photolyses of Metal Compounds: Cupric Chloride in Organic Media. J. Am. Chem. Soc. 1962, 84, 11, 2121–2127